This invention relates generally to the fields of molecular biology and pharmaceutical methods of treatment. More particularly, the invention relates to compounds, compositions, and methods for modulating p38 MAP kinase activity.
Activation of members of the mitogen-activated protein kinase (MAPK) family represents one of the major mechanisms used by eukaryotic cells to transduce extracellular signals into cellular response (J. Blenis, Proc Natl Acad Sci USA (1993) 90:5889). One member of this kinase family, p38, has been implicated in signaling pathways used by biologically important stimuli including products of microbial pathogens, cytokines, UV light and increased extracellular osmolarity. Most recently p38 activation was found to be correlated with apoptosis in neuronal cells following withdrawal of nerve growth factor. The kinase p38 is activated by a subset of the known dual-specificity MAPK kinases (MEKs or MKKs); low molecular weight GTP-binding proteins RAC1 and Cdc42 have also been shown to play a role in regulating p38 activation by some stimuli. A complete understanding of how p38 activation regulates cellular responses requires identification of specific substrates for this enzyme.
We have now identified compounds that modulate the activity of p38.
One aspect of the invention is a compound of formula 1: 
wherein R1 is H, lower alkyl, orxe2x80x94OR5, where R5 is aryl, heterocyclyl, acyl, Nxe2x80x94R6-4-piperidinyl, Nxe2x80x94R6-4-piperidinylmethyl, or Nxe2x80x94R6-4-piperidinylethyl, where aryl and heterocyclyl are substituted with 1-3 R6 substituents selected from the group consisting of H, lower alkyl, lower alkenyl, OH, NO2, NH2, halo, trihalomethyl, xe2x80x94CN, SH, SO, SO2, SO3H, xe2x80x94OR, xe2x80x94COR, xe2x80x94COOR, xe2x80x94CONHR, xe2x80x94OCOR, and xe2x80x94NCOR, where R is lower alkyl; R2 is H or lower alkyl; R3 is aryl substituted with 1-3 R6 substituents as set forth above; R4 is H, lower alkyl, aryl, aralkyl, xe2x80x94OH, xe2x80x94NH2, xe2x80x94NHR, or xe2x80x94OR, where R is lower alkyl or aryl-lower alkyl; and A is xe2x80x94CHxe2x80x94 or xe2x80x94Nxe2x80x94; and pharmaceutically acceptable salts thereof.
Another aspect of the invention is a pharmaceutical composition, comprising a compound of formula 1 and a pharmaceutically acceptable excipient.
Another aspect of the invention is a method for modulating p38 activity in a cell, comprising administering an effective amount of a compound of formula 1 to said cell.
Definitions:
xe2x80x9cCompound of formula 1xe2x80x9d refers to compounds having the structure 
wherein R1 is H, lower alkyl, or OR5, where R5 is aryl, heterocyclyl, acyl, Nxe2x80x94R6-4-piperidinyl, Nxe2x80x94R6-4-piperidinylmethyl, or Nxe2x80x94R6-4-piperidinylethyl, where aryl and heterocyclyl are substituted with 1-3 R6 substituents selected from the group consisting of H, lower alkyl, lower alkenyl, OH, NO2, NH2, halo, trihalomethyl, xe2x80x94CN, SH, SO, SO2, SO3H, xe2x80x94OR, xe2x80x94COR, xe2x80x94COOR, xe2x80x94CONHR, xe2x80x94OCOR, and xe2x80x94NCOR, where R is lower alkyl; R2 is H or lower alkyl; R3 is aryl substituted with 1-3 R6 substituents as set forth above; R4 is H, lower alkyl, aryl, aralkyl, xe2x80x94OH, xe2x80x94NH2, xe2x80x94NHR, or xe2x80x94OR, where R is lower alkyl or aryl-lower alkyl; and A is xe2x80x94CHxe2x80x94 or xe2x80x94Nxe2x80x94; and pharmaceutically acceptable salts and esters thereof. It should be noted that the two amidine nitrogen atoms can tautomerize if R2 is H, in which case either R1 or R3 can be bound to the imide nitrogen (the nitrogen double-bonded to carbon). Thus, no absolute geometry is indicated or intended around the R1xe2x80x94Nxe2x95x90C bond.
The term xe2x80x9clower alkylxe2x80x9d refers to radicals containing carbon and hydrogen, without unsaturation, having from one to six carbon atoms, inclusive. Lower alkyl radicals can be straight or branched. Exemplary lower alkyl radicals include, without limitation, methyl, ethyl, propyl, isopropyl, hexyl, t-butyl, and the like. The term xe2x80x9clower alkenylxe2x80x9d refers to a hydrocarbon radical having 2-6 carbon atoms, and at least one double bond. Exemplary lower alkenyl radicals include, without limitation, vinyl, propenyl, butenyl, and the like.
The term xe2x80x9carylxe2x80x9d refers to an aromatic carbocyclic or heterocyclic moiety, having one, two, or three rings. Exemplary aryl radicals include, without limitation, phenyl, naphthyl, pyridyl, pyrimidyl, triazyl, quinazolinyl, pyranyl, thiazolyl, and the like. The terms xe2x80x9caralkylxe2x80x9d and xe2x80x9caryl-lower alkylxe2x80x9d refer to an aryl moiety joined to a lower alkyl moiety, for example benzyl, phenethyl, 2-phenylpropyl, naphthylmethyl, and the like.
The term xe2x80x9chaloxe2x80x9d refers to fluoro, chloro, bromo, and iodo.
The term xe2x80x9cleaving groupxe2x80x9d refers to a radical that is easily displaced by a nucleophile in an SN2 displacement reaction. Exemplary leaving groups include, without limitation, sulfonates such as tosylate and mesylate, silanes such as t-butyl-dimethylsilane, halogens such as bromo and chloro, and the like.
The term xe2x80x9cpharmaceutically acceptable salts and estersxe2x80x9d refers to derivatives of compounds of formula 1 obtained by addition of an acid or base to the compound, or condensation with an alcohol or carboxylic acid to form an ester. In either case, the acid, base, alcohol, or carboxylic acid must not be unacceptably toxic at the concentrations at which the compound is administered. Suitable acids include, without limitation, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid or phosphoric acid; organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid and the like.
The term xe2x80x9cmodulatexe2x80x9d as used herein refers to an alteration in p38 MAP kinase activity, and includes both increases and decreases in activity. Modulation of activity can occur as the result of direct interaction of a compound with p38, interaction with another compound or protein that affects p38 activity directly or indirectly, or by altering the expression of p38 or of a protein that interacts with p38 directly or indirectly.
General Method:
Compounds of the invention are prepared by any suitable synthetic scheme. For example, an intermediate of formula A can be treated with an alcohol (Rxe2x80x94OH) and NaH or Naxe2x80x94OR to form intermediate B. 
Intermediate B can then be treated with an intermediate of the form R1xe2x80x94L (where L is a leaving group) in the presence of a strong, hindered base to provide an intermediate of formula C. This intermediate is then treated with an amine of the formula HNR2R3 under acidic or basic catalysis to provide the compound of formula 1. 
Compounds of the invention can be tested for activity by any available assay suitable for determining p38 activity or interference therewith.
Compounds of the invention can be administered to a subject, or can be applied directly to cells, for example in a cell culture. If administered to a cell culture, the compound is preferably first suspended or dissolved in a suitable carrier. Suitable carriers include, without limitation, water, saline solution, dimethylsulfoxide (DMSO) and solutions thereof, cell culture media, and the like.
Useful pharmaceutical carriers for the preparation of the pharmaceutical compositions hereof can be solids or liquids. Thus, the compositions can take the form of tablets, pills, capsules, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, and the like. Carriers can be selected from the various oils, including those of petroleum, animal, vegetable or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water, saline, aqueous dextrose, and glycols are preferred liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. Other suitable pharmaceutical carriers and their formulations are described in xe2x80x9cRemington""s Pharmaceutical Sciencesxe2x80x9d by E. W. Martin.
A compound of formula 1 or a pharmaceutical composition containing same is administered via any of the usual and acceptable methods known in the art, either singly or in combination with another compound or compounds of the present invention or other pharmaceutical agents. These compounds or compositions can thus be administered orally, systemically (e.g., transdermally, intranasally or by suppository) or parenterally (e.g., intramuscularly, subcutaneously and intravenously), and can be administered either in the form of solid or liquid dosages including tablets, solutions, suspensions, aerosols, and the like, as discussed in more detail above. It is preferred to administer compounds of formula 1 orally. The formulation can be administered in a single unit dosage form for continuous treatment or in a single unit dosage form ad libitum when relief of symptoms is specifically required.